It has become increasingly clear that the DNA damage response network extends beyond the canonical ATM/Chk2 and ATR/Chk1 signaling modules, to include connections to pathways as diverse as those involving PI 3-kinase/AKT, IKK/NFκB, and various MAP Kinases (MAPKs) [63
]. Multiple lines of evidence suggest an increasingly important role for the p38MAPK pathway and its downstream effector kinase MK2.
The p38MAPK/MK2 signaling complex is considered to be a general stress response pathway, which is activated in response to a variety of extrinsic and intrinsic stimuli including osmotic stress, heat shock, various toxins, UV and ionizing radiation, reactive oxygen species (ROS), cytokines, loss of centrosome integrity and DNA damage [67
]. p38MAPK activation drives a plethora of changes in transcription, protein synthesis, cell surface receptor expression, and cytoskeletal structure, ultimately affecting cell survival and apoptosis.
There are 4 p38MAPK isoforms denoted α, β, γ, and δ [67
]. Splice variants exist for p38α and β, giving rise to a total of six p38MAPK isoforms [68
]. p38α was first identified as a tyrosine-phosphorylated protein in extracts of LPS-treated macrophages and was subsequently shown to have significant homology with the yeast stress kinase HOG1 [68
]. Additional members of the p38MAPK family were cloned by homology. p38α appears to be ubiquitously expressed, with high levels in leukocytes, liver, spleen, bone marrow, thyroid, and placenta. In contrast, p38β shows some specificity for brain and heart, while p38γ is expressed at highest levels in skeletal muscle. p38δ has been reported to be expressed at highest levels in the lung, kidney, gut, salivary gland and endocrine organs such as the testis, ovary, adrenal-, and pituitary gland [68
]. Intriguingly, p38α exists in a stable complex with its downstream substrate, the kinase MK2 [69
], and the stability of p38α has been reported to depend on the presence of MK2. Gaestel and colleagues reported modestly reduced levels of p38MAPK in MK2-deficient mice [73
]. Whether similar complexes exist with p38β, γ, or δ is less clear. As with all known MAPKs, p38MAPKs are activated by dual phosphorylation on a T-X-Y motif in the activation loop through the action of MAP kinase kinases (MAPKKs, or MKKs). MAPKKs in turn are activated by a plethora of different MAP kinase kinase kinases (MAPKKKs, or MEKKs).
Many upstream MAPKKKs are known to participate in activation of the p38MAPK cascade including MEKK1/4, ASK1, and TAO kinases, depending on the particular type of initiating stimulus, which the cell is responding to. Using functionally p53-deficient HeLa cells, Cobb and colleagues showed that the MAPKKK TAO, activated p38α in an ATM-dependent manner in response to UV, IR and hydroxyurea treatment [74
]. Davis and colleagues, on the basis of overexpression studies, mouse knock-outs, and siRNA experiments, have shown that the MAPKKs MKK3 and 6 are the major upstream activators of p38α, β2, and γ in response to hyperosmolar stress and signaling by the pro-inflammatory cytokines TNFα and IL-1, while MKK4 has a redundant role along with MKK3 and 6 in p38MAPK activation by UV-irradiation [75
]. MKK4 is a known JNK MAPKK, suggesting that it may function as a signaling hub to integrate JNK and p38MAPK signaling [77
]. The upstream MAPKK activators of p38MAPK in response to genotoxic agents other than UV has not been reported.
Among the known substrates of p38MAPK are a number of transcription factors, including ATF1/2/6, MEF2A/C, p53, SAP1, STAT1, Gadd153 and Max, as well as the MAPK activated protein kinases MSK1 and 2, MNK1 and 2 and MK2, 3, and 5 (for detailed reviews see [67
Over the last decade a number of observations have been published that point to a critical role for the p38MAPK module as an integral part of the DNA damage response network. Besides UV, p38α and β have been shown to be activated by other, more DNA damage-specific agents, such as cisplatin, doxorubicin and temozolomide [6
]. Hirose et al.
] observed a p38MAPK-dependent G2
/M arrest following temozolomiode exposure in a mismatch repair-proficient human glioma cell line. p38MAPK signaling was associated with nuclear inactivation of Cdc25C, and RNAi-mediated knock down of p38α or pharmacologic inhibition of p38α and β reversed these effects. Mikhailov et al.
] were able to show that p38MAPK signaling is activated upon treatment of PtK1
cells with topoisomerse II inhibitors, and resulted in a late G2
/early prophase (so-called ‘antephase’) delay prior to mitotic entry. This delay could be overridden upon pharmacological inhibition of p38α and β. In this cell type, p38α and β were not required for normal mitotic progression in the absence of topoisomerase inhibition, or for the spindle assembly checkpoint. Although not a direct genotoxin, Sun et al., examined H-rasV12
oncogene-induced replicative stress, and found that p38MAPK mediated activation of MK5 is required for H-rasV12
-induced senescence in a murine model of DMBA-induced skin carcinogenesis. Induction of p38MAPK-dependent senescence was mediated by MK5-induced phosphorylation of p53 on Ser-37 [82
], and suggests that the p38MAPK pathway may function as a tumor suppressor in this context. Further insight into the DSB-mediated activation of p38MAPK came from the study of VDJ recombination in DN3 thymocytes. During T-lymphocyte maturation, cells undergo physiological induction of DSBs by RAG recombinase, followed by DSB repair in a DNA-PKcs-dependent manner. Pedraza-Alva et al
] used wild-type (WT) DN3 thymocytes, as well as Rag−/−
and Scid DN3s. WT cells were found to activate p38MAPK when DSBs were generated during VDJ recombination, and concomitantly underwent a G2
/M arrest. While Rag−/−
cells do not accumulate unresolved DSBs, Scid cells do so, due to a lack of DNA-PK activity, which is mutated on the Scid background. Since DNA-PK is a key component of the NHEJ repair machinery, Scid thymocytes are unable to repair the Rag induced DSBs. When the authors compared levels of activated p38MAPK in Rag−/−
and Scid thymocytes, they found increased levels of active p38MAPK only in Scid cells. The authors further describe the accumulation of both phosho-p38MAPK and phospho-p53 in Rag−/−
thymocytes expressing constitutively active MKK6, and suggested that p38MAPK, acting through p53, was responsible for the G2
/M checkpoint observed in these cells. Phosphorylation of p53 on Ser-18 and Ser-389 (corresponding to human Ser-15 and Ser-392) depended on p38MAPK and was abolished by the addition of the p38MAPK inhibitor SB203580. This study indicated that the p38MAPK pathway is activated in response to the accumulation of DSBs independently of DNA-PK, and may be involved in a p53-dependent G2
Kurosu and colleagues examined the role of p38MAPK in Burkitt’s lymphoma cells treated with the topoisomerase II inhibitor etoposide. This DNA damage-specific approach revealed that p38MAPK is activated following induction of DNA DSBs after topoisomerase II inhibition. In subsequent experiments using pharmacological inhibition of p38MAPK, as well as inducible expression of a dominant negative p38MAPK mutant, they were able to demonstrate that p38MAPK is required for an etoposide induced G2
/M arrest and that abrogation of this checkpoint resulted in increased apoptosis [84
Fornace and colleagues made the important observation that p38α and β signaling is necessary for the initiation of a G2
/M arrest after low-dose UV-irradiation, with p38α playing a more prominent role. Application of the specific p38MAPK inhibitor SB202190, or reduction of p38α and/or β levels using antisense oligonucleotides reversed this effect resulting in sustained mitotic activity for the first several hours after irradiation, although the mitotic index fell to similar levels as those observed in control cells 8 hrs later [85
]. This group further reported that p38MAPK-containing immunoprecipitates contained a kinase activity capable of directly phosphorylating Cdc25B and C to generate critical 14-3-3 binding sites, an effect which was interpreted as evidence that p38MAPK could directly generate 14-3-3-binding sites on Cdc25B/C. Ben-Levi et al.
], however, demonstrated that p38α forms a tight nuclear complex with its downstream substrate MK2 and that upon activation of p38MAPK in this complex, p38MAPK phosphorylates and activates MK2. This phosphorylation is essential for the nuclear export of the p38MAPK/MK2 complex in response to arsenite, and presumably other activating stimuli [69
]. Based on a long–standing interest in p38MAPK and its substrates, we determined the optimal sequence motifs phosphorylated by p38α and its downstream effector kinase MK2 [6
] using oriented peptide library screening. The optimal p38MAPK motif requires the presence of a Pro immediately C-terminal to the phospho-acceptor Ser or Thr residue, and does not conform to the critical 14-3-3- binding sites on Cdc25B or C. In contrast, the optimal phosphorylation motif found for MK2, ([L/I/F]-X-R
, where [S/T] denotes the phosphoacceptor residue and
indicates a hydrophobic amino acid, perfectly matches the known pSer-323 14-3-3 binding site in Cdc25B, as well as pSer-216 14-3-3 binding site in Cdc25C [6
]. Manke et al.
went on to demonstrate that recombinant MK2 from bacteria directly phosphorylated Cdc25B on Ser-323, thereby generating a 14-3-3 binding site on this molecule, and that RNAi-mediated knockdown of MK2 abolished the increase in 14-3-3 binding to cdc25B and C upon UV irradiation. Ducommun’s lab recently mapped the p38MAPK and MK2 phosphorylation sites on Cdc25B by mass spectrometry, providing independent confirmation that MK2 generates the Ser-323 binding site for 14-3-3 [86
]. Manke et al.
found that MK2-depleted cells were defective in both the G1
/S and G2
/M checkpoints after irradiation, rendering them more sensitive to UV-induced cell death as a consequence of mitotic catastrophe following checkpoint dysfunction. Together, these observations indicated that MK2 is the critical checkpoint effector that functions downstream of p38MAPK to arrest cell cycle progression in response to UV irradiation.
This role as a cell cycle regulator for MK2 appears to be highly conserved among eukaryotes. Lopez-Avilez et al.
] showed that overexpression of Srk1, the S. pombe
homologue of human MK2, caused a delay in mitotic entry while cells lacking Srk1 were shown to enter mitosis prematurely. These cell cycle regulatory effects resulted from Srk1 interaction with, and phosphorylation of, Cdc25 at sites at least partially identical to those phosphorylated by Chk1 and Cds1, the S. pombe
homologue of Chk2. Following Srk1-dependent phosphorylation, Cdc25 bound to Rad24, one of two S. pombe 14-3-3 proteins, leading to Cdc25 nuclear exclusion, stabilization, and catalytic inhibition, essentially recapitulating the identical MK2-Cdc25-14-3-3 pathway seen in mammalian cells. In contrast to mammalian cells, however, Srk1-deficient S. pombe
cells did not display increased sensitivity to UV irradiation, although depletion of Chk1 rendered them highly sensitive. Thus, in fission yeast, it appears that Srk1 functions during the normal G2
phase of an unperturbed cell cycle, but is not part of the UV-induced DNA damage cell cycle checkpoint response. Srk1 activation towards Cdc25 is controlled by Stk1, the S. pombe
homologue of p38MAPK, with which Srk1 forms a stable complex in the resting state [88
The budding yeast S. cerevisiae
contains two potential MK2 homologues, rck1
In an unbiased global screen of nearly 5,000 haploid deletion strains, Begley et al.
observed that loss of rck2
, resulted in increased UV sensitivity, indicating that this MK2 homologue is physiologically important for the cellular response to UV-induced damage in budding yeast [89
Mammalian MK2, like the fission yeast homologue, may also control cell cycle progression in response to stimuli other than direct DNA damage. Huard et al
] recently reported that the viral protein R (VPR) of HIV, which induces a G2
arrest in lymphocytes to block clonal T-cell expansion, does so by activating MK2. Similar to our observations on UV-activation of MK2, this VPR-activated form of MK2 was found to mediate phosphorylation of Cdc25C on the known Ser-216 14-3-3-binding site. Okamoto et al.
reported that HIV gp120, which induces a G1
arrest in neuronal stem cells, and may contribute to HIV-induced dementia, also functions through the activation of the p38MAPK/MK2 pathway. These HIV-related findings suggest that global cellular stresses from viral infection events trigger MK2-mediated cell cycle arrest, likely independently of damage to the DNA backbone or bases [91
When DNA is intentionally damaged by anti-cancer chemotherapeutic agents in mammalian cells, activation of the p38MAPK pathway appears to require ATM and ATR. Using ATM and ATR-deficient human cells as well as pharmacological inhibition of ATM and ATR, Reinhardt et al.
recently showed that the p38MAPK/MK2 module operates downstream of ATM and ATR in response to cisplatin, camptothecin and doxorubicin [7
]. Interestingly, p38MAPK/MK2 activation in response to UV was shown to be independent of ATM and ATR, suggesting that UV likely causes cellular lesions other than DNA damage that result in p38MAPK activation. Importantly, cisplatin- and doxorubicin-mediated activation of the p38MAPK/MK2 module was independent of Chk1, and conversely, Chk1 activation was independent of MK2 activity, indicating that the ATR/ATM-p38MAPK-MK2 pathway functions in parallel with the ATR-Chk1 pathway. The notion that MK2 operates in a synergistic parallel pathway to the classical checkpoint effectors Chk1 and Chk2 is further supported by the observation that S. cerevisiae
MK2 homologues rck1
can function as extragenic suppressors of S. pombe
cell cycle checkpoint mutations in chk1
, as well as mutations in rad1, rad9, rad17, and rad26
], reversing the sensitivity of these strains to UV and IR, and to the replication blocker hydroxyurea. The dependence on the canonical DNA damage response kinases ATM and ATR for p38MAPK/MK2 activation following genotoxic stress has now been confirmed independently [74
To further investigate whether there was a context-dependent role for MK2 in cell survival following genotoxic stress, Reinhardt et al.
recently investigated the effects of MK2 depletion in p53-proficient and p53-deficient MEFs [7
]. MK2 activity appeared to be dispensable for cellular survival following cisplatin or doxorubicin in p53-proficient cells. However, MK2 depletion in p53-deficient MEFs resulted in an increased sensitivity to both cisplatin and doxorubicin. This synthetic lethality between the prominent tumor suppressor p53 and MK2 was due to the inability of p53-deficient cells expressing MK2 shRNA to execute functional G1
/S and G2
cell cycle checkpoints following doxorubicin or cisplatin. Similar results were obtained when allograft tumors derived from p53-deficient, H-rasV12
-transformed MEFs were examined for chemosensitivity in a nude mouse model in vivo
. Knockdown of MK2 in these tumors resulted in dramatically increased sensitivity to systemic chemotherapeutic treatment with cisplatin or doxorubicin. Intriguingly, MK2-depleted tumors grew more rapidly than control tumors, indicating a potential role of MK2 as a negative regulator of the unperturbed cell cycle in tumor cells, and a potential tumor suppressor.
Although several groups have now confirmed members of the Cdc25 family as physiological MK2 substrates following genotoxic stress [6
], the question remains whether other checkpoint-relevant substrates for MK2 exist in vivo
. It is intriguing to speculate that a pool of substrates exists that is not
shared by Chk1 and MK2. For example, although both kinases recognize and phosphorylate the same optimal amino acid motif sequence, and both are present in the nucleus, active MK2 has been shown to translocate to the cytoplasm, in response to osmostic stress, arsenite exposure, and cytokine stimulation [69
]. It is therefore intriguing to speculate that MK2 might control a late cytoplasmic component of the DNA damage response. Among the known targets of MK2 following cytokine stimulation are a number of proteins involved in post-transcriptional regulation of mRNA and protein translation, including Ago2, a protein critical for the RNAi pathway [94
]. Could MK2-imposed control of mRNA metabolism, microRNA function, and protein translation also play a critical role in the DNA damage response? One important piece of evidence that hints at such a control mechanism is the result of a large-scale mass-spectrometry screen that sought to identify putative ATM and ATR substrates. This screen revealed that a substantial fraction of the putative substrates were proteins known to be involved in RNA metabolism [50
]. It is therefore tempting to speculate that the p38MAPK/MK2 complex might function by modulating the translation efficiency of a subset of critical mRNAs, which ultimately dictate cell fate following genotoxic stress. Hopefully future experiments will be able to support or refute this intriguing concept.